Intravenous administration of recombinant tPA remains the most beneficial proven intervention for emergency treatment of stroke. However, low reperfusion rates, short treatment time windows, and complications of hemorrhagic transformation and neurotoxicity comprise the major limitations for tPA clinically. In this application, we propose a translational plan to develop recombinant Annexin A2 protein (rA2) plus low-dose tPA as a combination thrombolytic therapy for acute ischemic stroke. We have 8 specific aims going from experiments in well-controlled rat embolic stroke models to preclinical studies for IND preparation and submission at the end of our 5-year project. 1. Dose-ranging studies for rA2 combined with low-dose tPA in a rat focal embolic stroke model. 2. Determine effects of optimal rA2 plus low-dose tPA combination in long term neurological outcomes. 3. Quantify recanalization/reperfusion efficacy and replicate neuroprotection in 2nd research site. 4. Determine the therapeutic time window of rA2 plus low-dose tPA. 5. Examine efficacy of rA2-low-dose-tPA combination in female and aged rat stroke models. 6. Examine efficacy of rA2-low-dose-tPA combination in hypertensive and diabetic rat stroke models. 7. Determine pharmacokinetic and toxicity profiles of rA2 plus low-dose tPA as a potential stroke therapeutic. 8. Submit IND application for the optimal combination dose of rA2 plus tPA based on efficacy, pharmacokinetic and toxicity profiles. In this U01, we are developing a drug based on a proven approach, i.e. thrombolysis and reperfusion. All we seek to do is to make tPA work better. Our goals are straightforward. Our milestones are based on quantitative go-no-go criteria. To be clinically relevant, the drug must improve long-term neurological function. It must actually recanalize like it's supposed to. it must work not just in healthy young males, but also in female, and animals with stroke risk factors (hypertension, aged). It must be safe. And of course, most importantly, it must be better than the standard tPA dosing. Our pilot data are promising. Our endpoints and milestones are directly related with what will be pursued in a clinical trial, i.e. neurological outcomes and imaging. If successful, this translational UOI prgram may lead us to a powerful new approach for stroke therapy. PUBLIC HEALTH RELEVANCE: Intravenous administration of tPA is the only FDA-approved emergency treatment for ischemic stroke. But shortcomings of brain bleeding risk, low efficacy and toxic side effects limit this application. Here we propose a preclinical program to develop a novel combination therapy of annexin A2 plus tPA that may make stroke thrombolysis safer and more efficacious.